Genomic Profile of Copy Number Variants on the Short Arm of Human

ARTICLE Genomic proﬁle of copy number variants on the short arm of human chromosome 8

Shihui Yu*,1, Stephanie Fiedler1, Andrew Stegner1 and William D Graf2

We evaluated 966 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization and found 10 individuals with pathogenic copy number variants (CNVs) on the short arm of chromosome 8 (8p), representing approximately 1% of the patients analyzed. Two patients with 8p terminal deletion associated with interstitial inverted duplication (inv dup del(8p)) had different mechanisms leading to the formation of a dicentric intermediate during meiosis. Three probands carried an identical B5.0 Mb interstitial duplication of chromosome 8p23.1. Four possible hotspots within 8p were observed at nucleotide coordinates of B10.45, 24.32–24.82, 32.19–32.77, and 38.94–39.72 Mb involving the formation of recurrent genomic rearrangements. Other CNVs with deletion- or duplication- speciﬁc start or stop coordinates on the 8p provide useful information for exploring the basic mechanisms of complex structural rearrangements in the human genome. European Journal of Human Genetics (2010) 18, 1114–1120; doi:10.1038/ejhg.2010.66; published online 12 May 2010

Keywords: microarray-based comparative genomic hybridization; short arm of chromosome 8; copy number variant; genomic disorders

INTRODUCTION MATERIALS AND METHODS The short arm of human chromosome 8 (8p) spans about 44 million Specimen acquisition base pairs containing 484 annotated genes (NCBI Build 36.3 of The DNA samples used for this research study came from 966 consecutive the human genome).1 Point mutations in more than 50 genes on pediatric patients referred for genome-wide screen testing by aCGH in our the 8p are associated with various genetic disorders and diseases laboratory. Each patient was evaluated because of one or more of the following (http://www.ncbi.nlm.nih.gov/omim). categorical clinical findings: global developmental delay, autism, dysmorphism, 8p is especially prone to various genomic rearrangements mainly seizures, or multiple congenital anomalies. The study protocol was approved by the institutional review board of Children’s Mercy Hospitals and Clinics. because of the existence of the two olfactory receptor gene clusters (REPD and REPP) flanking an B5 Mb region of 8p23.1.2–6 These Testing using chromosome banding analysis (GTG banding) and REPD- and/or REPP-related 8p genomic rearrangements include fluorescence in situ hybridization technique (1) the 8p23.1 deletion or duplication between REPD and REPP,6–9 8,10 Standard GTG banding analyses were performed on the peripheral blood (2) the 8p23.1 paracentric inversion between REPD and REPP, previously (patients 2, 3, 4, 6, 8, and 10) or concurrently (patients 1, 5 and 7, (3) the pericentric inversion (inv(8)(p23.1q22.1)) and recombinant 8, 9), and on the cultured skin fibroblast cells (patient 10). Fluorescence in situ 11 chromosome 8 (rec(8)dup(8q)inv(8)(p23.1q22.1)), (4) the 8p inter- hybridization (FISH) tests were performed for patient 3 using an 8p sub- stitial inverted duplication with associated terminal deletion (inv dup telomeric probe (AFM 197XG5), and for patient 10 using a chromosome 8 del(8p)),5,6,8,10,12–24 (5) the 8p translocations involving the 8p23.1,25,26 centromeric probe (CEP 8) as recommended by the manufacturer (Abbott and (6) different types of supernumerary chromosome 8 (SMC(8)) Molecular Inc., Des Plaines, IL, USA). involving the breakpoints within 8p23.1.4,27 In addition to these defined 8p genomic abnormalities, other pathogenic genomic changes Testing using microarray-based comparative genomic hybridization have been identified,28–30 whereas numerous genomic imbalances on The aCGH platform used in this study is the Agilent Human Genome 8p are still described as copy number variants (CNVs) of unknown Microarray Kit 244K, a genome-wide screening platform, containing 10 960 distinct 60-mer oligonucleotide probes on chromosome 8, with average probe clinical significance or CNVs without apparent clinical significance spacing of 13.3 kb (Agilent Technologies, Santa Clara, CA, USA). All aCGH (benign CNVs) (http://projects.tcag.ca/variation). tests were performed and analyzed following the protocols described In this study, we describe a comprehensive CNV profile of 8p previously.31 derived from the tests of a large number of pediatric patients with diverse clinical phenotypes using a high-resolution microarray- aCGH result verification and parental follow-up based comparative genomic hybridization (aCGH) platform, and The GTG banding analysis results for patients 1, 2, 3, 7, and 10 and FISH results discuss plausible mechanisms for the formation of these genomic for patients 2, 3, and 10 were used for verification of the abnormal aCGH rearrangements. findings on 8p (Table 1; Supplementary Figure S1). Quantitative real-time PCR

1Department of Pathology, Children’s Mercy Hospitals and Clinics and University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA; 2Section of Neurology, Children’s Mercy Hospitals and Clinics and University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA *Correspondence: Dr S Yu, Department of Pathology, Children’s Mercy Hospitals and Clinics and University of Missouri-Kansas City School of Medicine, 2401 Gillham Road, Kansas City, MO 64108, USA. Tel: +1 816 346 1303; Fax: +1 816 802 1204; E-mail: [email protected] Received 17 November 2009; revised 1 April 2010; accepted 1 April 2010; published online 12 May 2010 Genomic proﬁle of CNVs on the short arm of 8p SYuet al 1115

Table 1 Pathogenic CNVs on the short arm of chromosome 8

Chromosome and FISH Start Stop Verification for Patient findings aCGH findings coordinate (bp) coordinate (bp) Size (bp) aCGH findings Parents availability, and test findings

1 46,XY,del(8)(p23.1) [C] 8p23.3p23.1Â1 1 10 453 863 10 453 862 GTG banding Both parents, normal findings by GTG banding 2 46,XY,add(8)(p12).ish 8p23.3p23.1Â1 1 6929891 6929890 GTG banding, Both parents, normal findings add(8)(P12)(wcp8+) [R] 8p23.1p21.2Â3 12 601 939 24 823 981 12 222 042 FISH and by GTG banding 8q24.3Â3 145 084 438 146 265 046 1180608 qPCR 3 46,XX,add(8)(p23.1).ish 8p23.3p23.1Â1 1 6913476 6913475 GTG banding, Both parents, normal findings add(8)(p23.1)(8pterÀ)[R] 8p23.1p21.2Â3 7358840 25 707 713 18 348 873 FISH and by GTG banding and qPCR qPCR 4 46,XX [R] 8p23.1Â3 7 256 029 11 898 119 4 642 090 qPCR Inherited from mother; father NA 5 46,XY,dup(8)(p23.1p23.2) [C] 8p23.1Â3 7 256 029 12 512 055 5 256 026 qPCR Both parents, normal findings by qPCR 6 46,XY,var(8)(p23.1p23.2) [R] 8p23.1Â3 7 358 840 11 904 101 4 545 261 qPCR Mother normal by qPCR; father NA 7 46,XY,del(8)(p12p22) [C] 8p22p12Â1 18 375 250 32 765 636 14 390 386 GTG banding Both parents, normal findings by GTG banding 8 46,XX,t(8;18;14) 2q35q36.1Â1 217 249 378 222 041 732 4792354 qPCR Both parents, normal findings (p21.3;q23.1; q24.1) [R] 4q35.2Â1 188 428 673 189 965 537 1536864 by GTG banding and qPCR 8p21.3p21.2Â1 22 680 632 24 395 832 1715200 14q22.2Â1 54 012 870 54 614 836 601 966 9 46,XY [C] 8p12Â1 32 189 595 35 280 851 3 091 256 qPCR Both parents, inherited from father 10 47,XY,+mar.ish mar(8) 8p11.23q11.1Â3 38 944 542 47 575 850 8 631 308 GTG banding Both parents, normal findings (D8Z2+) from blood; and FISH by GTG banding 47,XY,+mar[20]/46,XY[7] from skin [R]

Abbreviations: C, concurrent chromosome analysis; R, retrospective chromosome analysis; Dup/Del, duplication/deletion; NA, not available.

(qPCR) were performed as previously described for verification of some and 3 have an B6.90 Mb terminal deletion of 8p with the breakpoints abnormal aCGH findings on 8p using different test primers targeting genes residing within the REPD region. The distal breakpoints of the within corresponding abnormal regions (RECQL4 for patient 1, GATA4 gene 8p23.1p21.2 duplications reside within REPP in patient 2 and REPD for patients 2-6, DLC1 gene for patient 3, NKX3 gene for patient 8, and MAK16 in patient 3, respectively. Three patients (4, 5, and 6) have an B5.0 Mb 32 for patient 9 (Table 1; Supplementary Table S1)). GTG banding, FISH, or interstitial duplication of 8p23.1 flanked by REPD and REPP (Figure 1; qPCR was also carried out for available corresponding parental follow-up Supplementary Figure S2b). The proximal breakpoint of the 14.39 Mb studies in this study (Table 1). deletion in patient 7, and the distal breakpoint of the 3.09 Mb deletion in patient 9 all reside within Neuregulin 1 (NRG1) gene within the RESULTS 8p11.23 region (Figure 1; Supplementary Figure S2c). aCGH testing Results from GTG banding and FISH testing identified four cryptic de novo deletions in four chromosomes in The GTG banding and FISH testing results are summarized in Table 1. patient 8 (Supplementary Figure S1c), including one of them with Of the 10 patients with abnormal aCGH results, 2 showed normal involvement of 8p21.3p21.2 (Figure 1; Supplementary Figure S2c). karyotypes (patients 4 and 9) and the remaining patients appeared to Patient 10 has a de novo mosaic pericentric SMC(8) (47,XY,+mar[20]/ have chromosomal rearrangements (Table 1). Images from GTG 46,XY[7]) (Supplementary Figure S1d). aCGH testing in this banding and/or FISH testing for patients 2, 3, and 10 are presented patient identified an 8.63 Mb duplication of 8p11.23q11.1 (Figure 1; in Supplementary Figure S1. Supplementary Figure S2c).

Pathogenic CNVs on 8p identified by aCGH-244K Verification of abnormal aCGH results in probands and Pathogenic 8p CNVs were found in 10 individuals (patients 1–10) in parental follow-up studies our study, representing B1% of the patients analyzed (Figure 1; The abnormal aCGH findings were verified in patients 1, 7, and 10 or Table 1). There are no other pathogenic genomic abnormalities except partially verified in patients 2 and 3 by GTG banding and FISH results in patients 2 and 8. Patient 1 has a 10.45 Mb terminal deletion with (Supplementary Figure S1a–c). The remaining abnormal aCGH proximal breakpoint residing between the MSRA and RP1L1 genes results were verified by qPCR (Table 1; Supplementary Table S1). within the REPD–REPP region of 8p23.1 (Figure 1; Supplementary Eighteen parental samples (eight couples and two single parents) Figure S2a). Two patients (patients 2 and 3) have inv dup del(8p) were available for determination of the inheritance pattern of the (Figure 1; Supplementary Figure S2a). Patient 2 has three genomic genomic abnormalities in their respective children (Table 1). The imbalances, a 6.93 Mb terminal deletion, a 12.22 Mb interstitial B4.6 Mb duplication of 8q23.1 in patient 4 was inherited from the duplication of 8p, and a 1.18 Mb terminal duplication of 8q. Patient mother; the B4.5 Mb duplication of 8q23.1 in patient 6 was negative 3 has two genomic imbalances, a 6.91 Mb terminal deletion, and an in the mother, but the father was not available for testing; the 18.35 Mb interstitial duplication of 8p. Within the 18.35 Mb interstitial B3.1 Mb deletion of 8p12 in patient 9 was inherited from the father. duplication, there is a 563 kb quintuplicated region, calculated by the The remaining tested parental samples showed negative results base-2 logarithm ratio of 1.33 in an aCGH analysis. Both patients 2 (Table 1).

European Journal of Human Genetics Genomic proﬁle of CNVs on the short arm of 8p SYuet al 1116

10 9 8 duplication 7 deletion 6 hotspot (HS) 5 4 3 2 1 HS

23.3 23.2 23.1 22 21.3 21.2 21.1 12 11.21

0M 10M 20M 30M 40M 50M OR-REPD OR-REPP UCSC segmental duplications

Figure 1 Locations of genomic abnormalities identiﬁed on chromosome 8p in this study. Numbers 1–10 represent the two patients with genomic abnormalities on chromosome 8p.

deletion- or duplication-speciﬁc benign CNVs were also observed in this study (Supplementary Table S2). Some start (Supplementary Table S4) or stop (Supplementary Table S5) breakpoints existed only in deleted CNVs and others occurred only in duplicated CNVs.

Clinical features in 10 patients with genomic abnormalities on 8p Clinical justiﬁcation for aCGH testing in these 10 patients with genomic abnormalities on 8p varied, but global development delay and dysmorphic features were the most common indications. Detailed description about the clinical ﬁndings in these patients is beyond the scope of this article, but is available in Supplementary Table S6.

DISCUSSION aCGH techniques have revolutionized the understanding of the human genome structure and are rapidly becoming new standard methods for clinical cytogenetics.33 These techniques have facilitated the identification of novel genomic disorders and precisely defined the breakpoint(s) of various genomic abnormalities. The findings in this study provide additional perspectives in viewing the underlying mechanisms causing genomic rearrangements on 8p.

REPD and REPP-mediated inv dup del(8p) Patients 2 and 3 in this study are postulated to have inv dup del(8p). Figure 2 Distribution of benign copy number variants (CNVs) across 8p. Three mechanisms may explain the origin of inv dup del(8p) Each dot represents a single benign CNV. (Figure 3) with detailed explanations referred to the recent reports.4,8,24,34 In brief, all three mechanisms involve the formation of a dicentric chromosome 8 (dic(8)) in meiosis I followed by Benign CNVs on 8p identiﬁed by aCGH-244K breakage of the dic(8) either during meiotic division or during early In addition to the pathogenic genomic abnormalities listed in Table 1, stages of embryonic development leading to the production of an inv 915 benign CNVs were observed on 8p in this study, 482 deletions and dup del(8p). However, the events causing the formation of a dic(8) 433 duplications (Figure 2; Supplementary Table S2). The majority of differ among the three mechanisms. these benign CNVs (93%) reside within three regions: 149 CNVs (50 Mechanism 1 involves a single parent (usually maternal) who dup/99 del) in region 1 (6 706 776–8 349 519 of the REPD region), 31 carries a paracentric inversion of 8p23.1 between REPD and REPP. CNVs (2 dup/41 del) in region 2 (11 903 701–12 630 984 of the REPP During meiosis I, the chromosome carrying the paracentric inversion region), and 655 CNVs (349 dup/306 del) in region 3 (39 323 211– pairs with its normal homologue by forming an inversion loop. 39 586 403) (Figure 2; Supplementary Table S3). Crossing-over and recombination within the loop create an unstable The 915 benign CNVs derived from the combination of 81 start and dic(8). Mechanism 2 involves the presence of inverted LCRs within the 85 stop breakpoints (Supplementary Tables S4 and S5). Although REPD or REPP of 8p. Partial folding of one homologue onto itself most benign CNV regions showed both deletion and duplication, with a recombination event between the inverted repeats leads to the

European Journal of Human Genetics Genomic proﬁle of CNVs on the short arm of 8p SYuet al 1117

E AAAAA A (Mechanism 1) DD

REPD REPP REPD REPP REPD REPP REPP REPP X region without deletion/duplication REPP REPD REPP REPD REPP REPD REPD REPD

D D D D D D D D Centromere E E E E E E E E

E A A A A (Mechanism 2) D D REPD REPD REPD REPP REPP region without REPP REPP X REPP deletion/duplication A A DDDD D D D D Centromere E E EE E E E E

D AA (Mechanism 3) REPP REPD REPD REPD REPD REPD REPD REPD REPP REPP REPP REPP REPP REPP REPP

D D D D D D D Centromere EEE E E E E

Figure 3 (a) Mechanism 1 in which the chromosome carrying the paracentric inversion between REPD and REPP pairs with its homologue by forming an inversion loop. Crossing-over and recombination within the loop create an unstable dicentric chromosome and an acentric fragment. The dicentric chromosome breaks outside the inverted region lead to the formation of a monocentric chromosome with a terminal deletion and an inverted duplication with a single copy region between the duplication. (b) Mechanism 2 in which the inverted LCRs within REPD or REPP in the same short arm of chromosome 8. Pairing and recombination between the inverted repeats on sister chromatids results in the formation of a dicentric chromosome and an acentric fragment. Breakage of the dicentric outside the inverted repeats leads to a monocentric chromosome with a terminal deletion and an inverted duplication with a single copy region between the duplication, which will be flanked by the inverted repeats. (c) Mechanism 3, which involves an initial premeiotic double-strand break of the two sister chromatids. Fusion of the broken ends results in a symmetric U-type reunion between the sister chromatids leading to the formation of a dicentric chromosome. Breakage distal to the fusion site outside the fusion region results in a monocentric chromosome with a terminal deletion and an inverted duplication without a single copy region between the duplication. formation of a dic(8). Mechanism 3 involves an initial premeiotic by mechanism 3.13,24 This finding is in contrast to the observation that double-strand break of the two sister chromatids of 8p. Fusion of the mechanism 3 is the most frequent mechanism for this type of genomic broken ends results in a symmetric U-type reunion between the sister rearrangements in all other chromosome arms.24 Although the inv chromatids leading to the formation of a dic(8). Both mechanisms dup del(8p) in patients 3 and two previously reported patients 1 and 2 produce inv dup del(8p) with a single copy region between the (Table 2) was caused by mechanism 3,13 there are several differences duplicated regions on the derivative chromosome 8, whereas mecha- among them. First, although there are no LCRs or other repeat nism 3 produces inv dup del(8p) without a single copy region between sequences around the breakpoints of the 3.9 Mb terminal deletion in the duplicated regions. The difference between mechanisms 1 and 2 is published case B and the 619 kb terminal deletion in published case C, that the single copy region between the duplications in mechanism 2 the breakpoint of the 6.91 Mb terminal deletion in patient 3 resides will be flanked by the inverted repeats. within the 8p REPD region, indicating that an unstable REPD region The inv dup del(8p) in patient 2 should be explained by mechanism could facilitate the initiation of a double-strand break of the two sister 2 because of the possible existence of inverted LCRs within the REPD chromatids and the formation of a symmetric U-type reunion or REPP on 8p, whereas mechanism 3 should explain the formation between the sister chromatids producing a dic(8). Second, both the of inv dup del(8p) in patient 3. The majority of the reported distal and proximal breakpoint sites of the 8p-duplicated regions in patients with inv dup del(8p) should be explained by mechanism these patients are different (Table 2). Third, the mechanisms to 25,6,8,10,12,14–23 with few exceptions (Table 2), which could be explained stabilize the broken chromosome ends are different, with telomere

European Journal of Human Genetics Genomic proﬁle of CNVs on the short arm of 8p SYuet al 1118

Table 2 Comparison of ﬁndings in patients 2 and 3 in this study with published case reports

Start Stop Size Formation of dicentric Patient/published case report Findings coordinate (Mb) coordinate (Mb) (Mb) chromosome Telomere capture

Patient 2 in this study 8pterp23.1Â1 0 6.93 6,93 NAHR (mechanism 2) with Yes? 8p23.1p21.2Â3 12.60 24.82 12,22 involvement of 8q24.3qter 145.08 146.26 1,18 REPD and REPP Patient 3 in this study 8pterp23.1Â1 0 6.91 6,91 U-type exchange (mechanism 3) No 8p23.1p21.2Â3 7.36 25.71 18,35 with involvement of REPD Case A in Buysse et al13 8pterp23.1Â1 0 6.90 6,90 NAHR (mechanism 2) with Yes 8p23.1p21.2Â3 12.63 16.97 3.40 involvement of REPD 8q24.13qter 125.34 146.25 20.90 and REPP Case B in Buysse et al13 8pterp23.1Â1 0 3.90 3,90 U-type exchange (mechanism 3) Yes 8p23.1p21.2Â3 4.48 24.31 19.83 without involvement of 8q24.13qter 117.93 146.25 28.30 REPD or REPP Case C in Rowe et al24 8pterp23.1Â1 0 0.61 0.61 U-type exchange (mechanism 3) No 8p23.1p21.2Â3 0.62 37.79 18,35 without involvement of REPD or REPP Cases D–H in 8pterp23.1Â1 8p23.1p21.2Â30 5.8–8.3 5.8–8.3 NAHR (mechanism 2) with No Shimokawa et al10 12.62 B39.72 26–31 involvement of REPD and REPP

Abbreviation: NAHR, nonallelic homologous misalignment and unequal recombination. For explanations of ‘mechanism 2’ and ‘mechanism 3’, see text.

capture in reported case of patient B, but not in reported case of This B5.0 Mb duplication of 8p23.1 is flanked by REPD and REPP, patient C and our patient 3 (Table 2). which share a high level of identity of complex genomic repeats Terminal deletions can result in severe genomic instability if not involving retroviral elements, olfactory repeat regions, and variable properly repaired.24 Thus, broken chromosome ends must acquire a copy numbers of defensin genes.4,7,8,42 The 8p23.1 duplication is likely new telomeric cap to remain structurally stable by ‘telomere healing,’ caused by intrachromosomal recombination between REPD and in which telomeric sequences can be acquired de novo,24 or by REPP within the 8p23.1 region through LCR-mediated nonallelic ‘telomere capture,’ in which broken chromosome obtains the telo- homologous misalignment and unequal recombination (NAHR) meric end of another chromosome.35–37 Alternatively, stabilization can during meiosis.6–9,43,44 occur through circularization of the inv dup del chromosome, leading The copy numbers of different types of defensin and olfactory to the formation of a ring chromosome.38 receptor genes in the REPD and REPP regions are highly variable, In addition to the 6.93 Mb 8p terminal deletion associated with a leading to cytogenetically visible euchromatic variants if the copies are 12.22 Mb interstitial duplication of 8p23.1p21.2, an additional sufficiently high.4,45,46 However, these euchromatic variants are indis- 1.18 Mb terminal duplication of 8q was identified by aCGH in patient tinguishable from the genuine 8p23.1 duplication under a light 2 (Figure 1; Table 1). It is possible that the broken chromosome end of microscope. For example, two of the three patients with 8p23.1 8p has been stabilized by telomere capture through an additional duplication in this study were initially reported as euchromatic rearrangement with distal 8q material, similar to three previously variants to the referring physicians. These results show the power of reported patients with inv dup del(8p).13,39 No additional tissue from aCGH with high resolution to reveal unexpected imbalances in our patient 2 was available for confirmation of this hypothesis using affected patients without specific clinical findings as well as the FISH methods. sensitivity to discriminate genuine 8p23.1 duplication from euchro- The distal end of the 563 kb de novo quintuplicated region in patient matic variants caused by high copies of defensin and olfactory receptor 3 exists just at the proximal end of the LCR–REPP region. It is likely genes. On the basis of the finding of three probands with 8p23.1 that the occurrence of this quintuplicated region may involve more duplication in this study, or a 0.31% detection rate, we suspect that the complex genomic rearrangements than we postulate here. However, 8p23.1 duplication may be more common than previously suspected.7 the mechanism causing this complexity is unknown. Rare genomic abnormalities reveal the possible existence of 8p23.1 duplication multiple hotspots leading to variable genomic rearrangements Although more than 10 patients having isolated 8p23.1 duplication on 8p have been reported,7,40–42 only 6 of them in four families have The specific genomic abnormalities of patients 1 and 7–10 in this been characterized by FISH or aCGH.7,42 Three probands (patients study have not been previously reported. However, these rare genomic 4–6) in this study were observed to have the 8p23.1 duplication abnormalities reveal the occurrence of several unstable regions leading (Table 1; Figure 1; Supplementary Figure S1b). The 8p23.1 duplication to recurrent genomic rearrangements on 8p. The proximal breakpoint in patient 4 was inherited from the affected mother. The 8p23.1 of the 10.45 Mb deletion within the REPD–REPP region of 8p23.1 in duplication in patient 5 occurred de novo. The inheritance patient 1 was recently reported in a transmitted 8p23.1p23.2 duplica- pattern for the duplication in patient 6 could not be determined tion in an individual with autism.47 because of negative results in the mother and the unavailability of Patient 8 in this study was found to have an apparently balanced the father. complex translocation (46,XX,t(8;18;14)(p21.3;q23.1;q24.1)), but

European Journal of Human Genetics Genomic profile of CNVs on the short arm of 8p SYuet al 1119 aCGH testing identified four cryptic de novo deletions of possible unknown, and it is possible that yet-to-be discovered mechanisms clinical significance (Table 1; Supplementary Figure S2c). The two prohibit the formation of certain deletions or duplications. deletions of 2q35q36.1 and 4q35.2 were not apparently associated with In summary, we report an integrated high-resolution CNV profile the complex translocation. Either the two deletions occur indepen- of human chromosome 8p derived from the analysis of 966 conse- dently without involvements of the complex translocation, or more cutive individuals using the aCGH-244K platform, and present complex chromosomal rearrangements with involvements of the two plausible mechanisms for the formation of inv dup del(8p) and deletions were missed by chromosome analysis. However, there is no 8p23.1 duplication. Several regions within 8p are proposed to be available specimen from this patient to further verify the complexity of hotspots leading to the formation of recurrent genomic rearrange- the chromosomal rearrangements. Such discoveries support the ments. CNVs with deletion- or duplication-specific start or stop hypothesis that some ‘balanced’ rearrangements include cryptic dele- breakpoints provide useful information for exploring underlying tions or more complex rearrangements,48 further emphasizing the mechanisms leading to the formation of complex structural rearran- benefit of high-resolution aCGH analysis. The proximal breakpoint of gements in the human genome. the 1.72 Mb deletion of 8p21.3p21.2 in this patient is flanked by repeat-rich sequences, indicating that this region could be an unstable CONFLICT OF INTEREST region for genomic arrangements. This hypothesis is supported by the The authors declare no conflict of interest. observation that the proximal breakpoints of the 12.82 Mb duplication in patient 2 and the 19.83 Mb duplication in reported case 2 reside within the same gene desert region (Table 2). In addition, the ACKNOWLEDGEMENTS proximal breakpoint of the duplication in reported case 4 appeared We thank Dr Holly H Ardinger for the critical review and comments on the to map to this region (Table 2).5 article. The NRG1 gene was disrupted in two of the ten abnormal patients (patients 7 and 9) in this study. Breakpoints of genomic rearrangements occurring in the NRG1 gene were observed in other reported cases with 8p genomic abnormalities,49,50 indicating that unknown 1 Nusbaum C, Mikkelsen TS, Zody MC et al: DNA sequence and analysis of human chromosome 8. Nature 2006; 439: 331–335. genomic structural features within the NRG1 gene may be susceptible 2 Vermeesch JR, Thoelen R, Salden I, Raes M, Matthijs G, Fryns JP: Mosaicism del(8p)/ to these various genomic rearrangements. Alternatively, patients with inv dup(8p) in a dysmorphic female infant: a mosaic formed by a meiotic error at the 8p disruption of this gene may be more likely to be investigated because OR gene and an independent terminal deletion event. JMedGenet2003; 40:e93. 3 Giorda R, Ciccone R, Gimelli G et al: Two classes of low-copy repeats comediate a new of the impact of the NRG1 gene on cardiac and neural development. recurrent rearrangement consisting of duplication at 8p23.1 and triplication at More than 60 cases with SMC(8) have been reported (http:// 8p23.2. Hum Mutat 2007; 28: 459–468. www.med.uni-jena.de/fish/sSMC/08.htm). Ten published cases with 4 Hollox EJ, Barber JC, Brookes AJ, Armour JA: Defensins and the dynamic genome: what we can learn from structural variation at human chromosome band 8p23.1. Genome SMC(8) include genomic material similar to patient 10 in this Res 2008; 18: 1686–1697. study.27,51 Although no annotated repeat sequences occur around 5 Cooke SL, Northup JK, Champaige NL et al: Molecular cytogenetic characterization of a unique and complex de novo 8p rearrangement. Am J Med Genet A 2008; 146A: the breakpoints of this SMC(8) in patient 10, the 8p breakpoint 1166–1172. (38.94 Mb) of this SMC(8) is adjacent to the proximal breakpoint 6 Shimokawa O, Miyake N, Yoshimura T et al: Molecular characterization of (B39.72 Mb) of the inverted duplicated regions in five reported cases del(8)(p23.1p23.1) in a case of congenital diaphragmatic hernia. Am J Med Genet A 10 2005; 136:49–51. with inv dup del(8p). This region appears to be extremely unstable 7 Barber JC, Maloney VK, Huang S et al: 8p23.1 duplication syndrome; a novel genomic because 72% (847 of 915) of the benign CNVs in this study carry condition with unexpected complexity revealed by array CGH. Eur J Hum Genet 2008; proximal and/or distal breakpoints within the region (Figure 2; 16:18–27. 8 Giglio S, Broman KW, Matsumoto N et al: Olfactory receptor-gene clusters, genomic- Table 2). On the basis of these rare genomic abnormalities on 8p, inversion polymorphisms, and common chromosome rearrangements. Am J Hum Genet we may conclude that the regions with nucleotide coordinates at 2001; 68: 874–883. B 9 Devriendt K, Matthijs G, Van Dael R et al: Delineation of the critical deletion region 10.45, 24.32–24.82, 32.19–32.77, and 38.94–39.72 Mb on 8p are for congenital heart defects, on chromosome 8p23.1. Am J Hum Genet 1999; 64: particularly susceptible to genomic rearrangements. 1119–1126. 10 Shimokawa O, Kurosawa K, Ida T et al: Molecular characterization of inv dup del(8p): analysis of five cases. Am J Med Genet A 2004; 128A: 133–137. Mechanisms leading to the formation of benign CNVs on 8p 11 Graw SL, Sample T, Bleskan J, Sujansky E, Patterson D: Cloning, sequencing, and Ninety-three percent of the benign CNVs on 8p identified in this analysis of inv8 chromosome breakpoints associated with recombinant 8 syndrome. study reside within three regions: the REPD and REPP regions of Am J Hum Genet 2000; 66: 1138–1144. 12 Weleber RG, Verma RS, Kimberling WJ, Fieger Jr HG, lubs HA: Duplication-deficiency 8p23.1, and the 8p11.23 region (Figure 2; Supplementary Table S3). of the short arm of chromosome 8 following artificial insemination. Ann Genet 1976; These results are concordant to the data documented in the Database 19: 241–247. 13 Buysse K, Antonacci F, Callewaert B et al: Unusual 8p inverted duplication deletion of Genomic Variants (http://projects.tcag.ca/variation/), reflecting the with telomere capture from 8q. Eur J Med Genet 2009; 52:31–36. extreme instability of these three regions. Variable copy numbers of 14 Floridia G, Piantanida M, Minelli A et al: The same molecular mechanism at the defensin and olfactory receptor genes within the REPD and REPP maternal meiosis I produces mono- and dicentric 8p duplications. Am J Hum Genet 4 1996; 58: 785–796. regions, and the large numbers of simple tandem repeats within the 15 Pabst B, Arslan-Kirchner M, Schmidtke J, Miller K: The application of region-specific 8p11.23 region should be the structural basis leading to the formation probes for the resolution of duplication 8p: a case report and a review of the literature. of these benign copy variations. Cytogenet Genome Res 2003; 103:3–7. 16 Dill FJ, Schertzer M, Sandercock J, Tischler B, Wood S: Inverted tandem duplication Different underlying mechanisms were proposed to explain the generates a duplication deficiency of chromosome 8p. Clin Genet 1987; 32: 109–113. formation of CNVs based on the structural features of the human 17 Barber JC, James RS, Patch C, Temple IK: Protelomeric sequences are deleted in 52–56 cases of short arm inverted duplication of chromosome 8. Am J Med Genet 1994; 50: genome. However, none of the proposed mechanisms can fully 296–299. explain the occurrence of some deletion- or duplication-specific start 18 Minelli A, Floridia G, Rossi E et al: D8S7 is consistently deleted in inverted duplica- or stop breakpoints of CNVs and some deletion- or duplication- tions of the short arm of chromosome 8 (inv dup 8p). Hum Genet 1993; 92: 391–396. 19 Guo WJ, Callif-Daley F, Zapata MC, Miller ME: Clinical and cytogenetic findings in specific CNVs observed in this study (Figure 2; Supplementary Tables seven cases of inverted duplication of 8p with evidence of a telomeric deletion using S2–S5). The actual mechanisms leading to this phenomenon remain fluorescence in situ hybridization. Am J Med Genet 1995; 58: 230–236.

European Journal of Human Genetics Genomic proﬁle of CNVs on the short arm of 8p SYuet al 1120

20 de Die-Smulders CE, Engelen JJ, Schrander-Stumpel CT et al: Inversion duplication of 38 Knijnenburg J, van Haeringen A, Hansson KB et al: Ring chromosome formation as a the short arm of chromosome 8: clinical data on seven patients and review of the novel escape mechanism in patients with inverted duplication and terminal deletion. literature. Am J Med Genet 1995; 59: 369–374. Eur J Hum Genet 2007; 15: 548–555. 21 Macmillin MD, Suri V, Lytle C, Krauss CM: Prenatal diagnosis of inverted duplicated 39 Kostiner DR, Nguyen H, Cox VA, Cotter PD: Stabilization of a terminal inversion 8p. Am J Med Genet 2000; 93:94–98. duplication of 8p by telomere capture from 18q. Cytogenet Genome Res 2002; 98: 22 Felbor U, Knotgen N, Schams G, Buwe A, Steinlein C, Schmid M: Mosaicism for an 9–12. ectopic NOR at 8pter and a complex rearrangement of chromosome 8 in a patient with 40 Kennedy SJ, Teebi AS, Adatia I, Teshima I: Inherited duplication, dup (8) severe psychomotor retardation. Cytogenet Genome Res 2004; 106: 55–60. (p23.1p23.1) pat, in a father and daughter with congenital heart defects. Am J Med 23 Zuffardi O, Bonaglia M, Ciccone R, Giorda R: Inverted duplications deletions: under- Genet 2001; 104:79–80. diagnosed rearrangements? Clin Genet 2009; 75: 505–513. 41 Tsai CH, Graw SL, McGavran L: 8p23 duplication reconsidered: is it a true euchromatic 24 Rowe LR, Lee JY, Rector L et al: U-type exchange is the most frequent mechanism for variant with no clinical manifestation? J Med Genet 2002; 39: 769–774. inverted duplication with terminal deletion rearrangements. J Med Genet 2009; 46: 42 Barber JC, Maloney V, Hollox EJ et al: Duplications and copy number variants 694–702. of 8p23.1 are cytogenetically indistinguishable but distinct at the molecular level. 25 Zollino M, Murdolo M, Marangi G et al: On the nosology and pathogenesis of Wolf– Eur J Hum Genet 2005; 13: 1131–1136. Hirschhorn syndrome: genotype–phenotype correlation analysis of 80 patients and 43 Slavotinek A, Lee SS, Davis R et al: Fryns syndrome phenotype caused by chromosome literature review. Am J Med Genet C Semin Med Genet 2008; 148C: 257–269. microdeletions at 15q26.2 and 8p23.1. J Med Genet 2005; 42: 730–736. 26 Ozkinay F, Kanit H, Onay H et al: Prenatal diagnosis of de novo unbalanced transloca- 44 Devriendt K, De Mars K, De Cock P, Gewillig M, Fryns JP: Terminal deletion in tion 8p;21q using subtelomeric probes. Genet Couns 2006; 17: 315–320. chromosome region 8p23.1-8pter in a child with features of velo-cardio-facial 27 Liehr T, Mrasek K, Weise A et al: Small supernumerary marker chromosomes – progress syndrome. Ann Genet 1995; 38: 228–230. towards a genotype–phenotype correlation. Cytogenet Genome Res 2006; 112:23–34. 45 Barber JC, Joyce CA, Collinson MN et al: Duplication of 8p23.1: a cytogenetic anomaly 28 Fellermann K, Stange DE, Schaeffeler E et al: A chromosome 8 gene-cluster poly- with no established clinical significance. J Med Genet 1998; 35: 491–496. morphism with low human beta-defensin 2 gene copy number predisposes to Crohn 46 Engelen JJ, Moog U, Evers JL, Dassen H, Albrechts JC, Hamers AJ: Duplication of disease of the colon. Am J Hum Genet 2006; 79: 439–448. chromosome region 8p23.1-p23.3: a benign variant? Am J Med Genet 2000; 91: 29 Cho SC, Yim SH, Yoo HK et al: Copy number variations associated with idiopathic 18–21. autism identified by whole-genome microarray-based comparative genomic hybridiza- 47 Glancy M, Barnicoat A, Vijeratnam R et al: Transmitted duplication of 8p23.1–8p23.2 tion. Psychiatr Genet 2009; 19:177–185. associated with speech delay, autism and learning difficulties. Eur J Hum Genet 2009; 30 Hollox EJ, Huffmeier U, Zeeuwen PL et al: Psoriasis is associated with increased beta- 17:37–43. defensin genomic copy number. Nat Genet 2008; 40:23–25. 48 Higgins AW, Alkuraya FS, Bosco AF et al: Characterization of apparently balanced 31 Yu S, Bittel DC, Kibiryeva N, Zwick DL, Cooley LD: Validation of the Agilent 244K chromosomal rearrangements from the developmental genome anatomy project. oligonucleotide array-based comparative genomic hybridization platform for clinical Am J Hum Genet 2008; 82: 712–722. cytogenetic diagnosis. Am J Clin Pathol 2009; 132: 349–360. 49 Willemsen MH, de Leeuw N, Pfundt R, de Vries BB, Kleefstra T: Clinical and molecular 32 Yu S, Kielt M, Stegner AL, Kibiryeva N, Bittel DC, Cooley LD: Quantitative real-time characterization of two patients with a 6.75 Mb overlapping deletion in 8p12p21 with polymerase chain reaction for the verification of genomic imbalances detected by two candidate loci for congenital heart defects. Eur J Med Genet 2009; 52:134–139. microarray-based comparative genomic hybridization. Genet Test Mol Biomarkers 50 Klopocki E, Fiebig B, Robinson P et al: A novel 8 Mb interstitial deletion of chromo- 2009; 13: 751–760. some 8p12–p21.2. Am J Med Genet A 2006; 140: 873–877. 33 Shinawi M, Cheung SW: The array CGH and its clinical applications. Drug Discov Today 51 Bettio D, Baldwin EL, Carrozzo R et al: Molecular cytogenetic and clinical findings in a 2008; 13: 760–770. patient with a small supernumerary r(8) mosaicism. Am J Med Genet A 2008; 146A: 34 Ciccone R, Mattina T, Giorda R et al: Inversion polymorphisms and non-contiguous 247–250. terminal deletions: the cause and the (unpredicted) effect of our genome architecture. 52 Makoff AJ, Flomen RH: Detailed analysis of 15q11–q14 sequence corrects errors and J Med Genet 2006; 43:e19. gaps in the public access sequence to fully reveal large segmental duplications at 35 Ballif BC, Gajecka M, Shaffer LG: Monosomy 1p36 breakpoints indicate repetitive DNA breakpoints for Prader–Willi, Angelman, and inv dup(15) syndromes. Genome Biol sequence elements may be involved in generating and/or stabilizing some terminal 2007; 8:R114. deletions. Chromosome Res 2004; 12: 133–141. 53 Lupski JR: Structural variation in the human genome. NEnglJMed2007; 356: 36 Ballif BC, Wakui K, Gajecka M, Shaffer LG: Translocation breakpoint mapping and 1169–1171. sequence analysis in three monosomy 1p36 subjects with der(1)t(1;1)(p36;q44) 54 Gu W, Zhang F, Lupski JR: Mechanisms for human genomic rearrangements. suggest mechanisms for telomere capture in stabilizing de novo terminal rearrange- Pathogenetics 2008; 1:4. ments. Hum Genet 2004; 114:198–206. 55 de Smith AJ, Walters RG, Coin LJ et al: Small deletion variants have stable breakpoints 37 Chabchoub E, Rodriguez L, Galan E et al: Molecular characterisation of a mosaicism commonly associated with alu elements. PLoS One 2008; 3:e3104. with a complex chromosome rearrangement: evidence for coincident chromosome 56 Hastings PJ, Ira G, Lupski JR: A microhomology-mediated break-induced replication healing by telomere capture and neo-telomere formation. J Med Genet 2007; 44: model for the origin of human copy number variation. PLoS Genet 2009; 5: 250–256. e1000327.

Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)

European Journal of Human Genetics